1. Technical Field
The present invention relates to an apparatus and method for obtaining images to reveal structures and properties of materials and more particularity, to an apparatus and method for obtaining images using coherent anti-stokes Raman scattering.
2. Related Art
Raman spectroscopy is a phenomenon that scatters light having a wavelength different from that of monochromatic light input from a sample on which the monochromatic light is irradiated. Recently, the Raman spectroscopy tracks a change of a vibration mode together with infrared spectroscopy, such that it has established an independent area of learning that reveals structures and properties of molecules.
Even though the Raman spectroscopy was studied earlier than the infrared spectroscopy due to characteristic using Raman scattering light having weak strength, it has been slower in development. As a result, the frequency in the use of the Raman spectroscopy has been low until now. However, with the advent of a laser having improved output strength, the Raman spectroscopy is being developed rapidly. As a result, the use of the Raman spectroscopy is currently of interest for various applications.
Meanwhile, a fluorescent microscope is considered as the epochal technology in the field of cell biology due to the help of the development of various fluorescence probes, confocal detection, and three-dimensional imaging through multi-photon excitation. The fluorescent microscope irradiates a light source at a wavelength that can excite fluorescent molecules, which is known as the fluorescence probes naturally existing in the sample or artificially injected into the sample, on the sample. At this time, the sample emits fluorescence by absorbing the excitation light source and is observed using a filter that selectively transmits fluorescence. The fluorescent microscope is advantageous in that it has higher resolution than a general optical microscope. However, since the fluorescent microscope uses the fluorescence probes, it has problems in that it changes the sample to be measured and causes a photobleaching phenomenon.
A microscope using coherent anti-stokes Raman scattering (CARS) that inputs fixed/variable laser lights to Raman active media and measures spectrums of anti-stokes light obtained by a combination thereof has been widely used on the grounds that it has high sensitivity and is not affected by the media generating fluorescence.
FIG. 1 is an energy band diagram for explaining a generation principle of a general CARS signal. The CARS is a four wave mixing process that generates anti-stokes light by interacting pump light, stokes light, and probe light with a sample and uses two laser beams having different frequencies. A first laser beam serves as the stokes light (ωs) and a second laser beam serves as the pump light (ωp) and the probe light (ωpro). In other words, the frequency of ωpro is identical with ωp. The electric field of the light is EP (ωp), ES (ωs), Epro (ωpro) (=EP (ωp)). If the laser beam is irradiated, electrons, which are in a ground state ν0=0 are first excited in a virtual state by the pump light (ωp) and most of the electrons are then transitioned into a level ν1=1 by the stokes light ωs. At this time, the inherent vibration frequency of the electrons becomes Ω by the resonant Raman scattering. After the electrons transitioned from ν0=0 to ν0=1 are back excited into a virtual state such as ωp+Ω by the probe light (ωpro), they emit the anti-stokes light having frequency ωas=2ωp−ωs that satisfies energy conservation, that is, CARS signals, and are transitioned to the level ν0=0. The CARS microscope analyzes materials by measuring the strength of the CARS signal.
Since the CARS microscope as described above has the same resolution as the confocal microscope but does not use emission of a pigment, it does not change the sample. Further, since the CARS microscope uses Raman that corresponds to a vibration level of chemical species, it has an advantage in that it can select the chemical species. Moreover, the CARS microscope can obtain a very large signal as compared to spontaneous Raman scattering and because it has anti-stokes frequency different from the frequencies used by the two laser beams, the signal can be easily separated by using a filter, etc.
However, a representative disadvantage of the coherent anti-stokes Raman scattering is a non-resonant background signal phenomenon caused due to two-photon electronic resonance, which has been studied by [M. D. Duncan, J. Reintjes, and T. J. Manuccia, “Scanning coherent anti-Stokes Raman microscope,” Opt. Lett. 7, 350-352, 1982]. It was found that any two-photon-enhanced background phenomenon exceeding the resonant vibration signal is generated due to the use of visible light. In a study by [A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82, 4142-4145, 1999.], it was found that two-photon electronic resonance is prevented and sensitivity increase, by using near-infrared light. Thereafter, with the development of the coherent anti-stokes Raman scattering spectrometer, several methods were used for reducing the non-resonant background signal.
However, even though there are many problems in the conventional technologies associated with the CARS, these technologies all detected the strength of the CARS signal and directly analyzed them. As a result, these technologies focused on the difference between the CARS signal and the non-resonant background signal, such that many problems occurred. In particular, a method focusing on the reduction of the non-resonant background signal weakens the strength of light to be detected, such that there is a need for a high-specification photo detector in order to detect the signal having weak strength without noise. Further, since the method directly analyzes the signal to be detected by the strength of signal, it has limitations in sensitivity, resolution, accuracy, etc.